RESEARCH ARTICLE

Occurrence and risk assessment of tetracycline antibiotics in soilfrom organic vegetable farms in a subtropical city, south China

Lei Xiang1 & Xiao-Lian Wu1 & Yuan-Neng Jiang1 & Qing-Yun Yan1 & Yan-Wen Li1 &Xian-Pei Huang1 & Quan-Ying Cai1 & Ce-Hui Mo1

Received: 16 June 2015 /Accepted: 16 March 2016 /Published online: 4 April 2016# Springer-Verlag Berlin Heidelberg 2016

Abstract This study investigated the occurrence of tetracy-cline antibiotics in soils from different organic vegetable farmsin Guangzhou, a subtropical city, South China and evaluatedtheir ecological risk. Four tetracycline compounds (oxytetra-cycline, tetracycline, chlortetracycline, and doxycycline) wereextracted ultrasonically from soil samples (n=69), with asolid-phase extraction cleanup, and were then measured byhigh-performance liquid chromatography–tandem mass spec-trometry (HPLC-MS/MS). The results showed that four com-pounds were detected in all samples, with the concentrationsof the individual compounds ranging from 0.04 to 184.8 μg/kg (dry weight). The concentrations of tetracycline com-pounds in the soils from different vegetable farms variedgreatly, but their patterns of distribution were similar.Doxycycline was the predominant compound with a meanof 21.87 μg/kg, followed by chlortetracycline. The concentra-tions of doxycycline and chlortetracycline in 7.46 % of thesamples were higher than the ecotoxic effect trigger value(100 μg/kg) set by the Steering Committee of Veterinary

International Committee on Harmonization. Additionally, theconcentrations of tetracyclines in greenhouse soils were sig-nificantly lower than those in open-field soils. Risk assess-ment based on single compound exposure showed that doxy-cycline could pose medium or high risks. Compared withother studies, the levels of tetracyclines in this study wererelatively low. The hypothesis that antibiotic residues in thesoil of organic farms fertilized with manure are higher than inthe soils of conventional farms was not supported in the areastudied due to the high levels of moisture, temperature, andmicrobial activity.

Keywords Organic vegetable farm . Soil . Antibiotics .

Tetracycline . Risk assessment . Subtropical area

Introduction

Antibiotics have been used extensively to treat disease andprotect animal health worldwide (Sarmah et al. 2006), andthe total amount of antibiotics used worldwide has been esti-mated to have reached ∼200,000 tons per year (Rehman et al.2013). Veterinary uses account for the majority of the totalantibiotics used. For example, in the USA and China,veterinary antibiotics approximately account for 70 and48 %, respectively, of the total consumption (Mellon et al.2001; Sassman and Lee 2005). Tetracycline antibiotics arecurrently among the most extensively used growth promotersand therapeutic drugs in animal agriculture (Cheng et al.2006; Sarmah et al. 2006; Kools et al. 2008). Antibioticscannot be completely absorbed in vivo, and about 80 % ofthe antibiotics used are excreted as parent compounds or me-tabolites via the waste of livestock in which the concentrationsof antibiotics ranged from dozens to thousands of mg/kg;(Jjemba 2002; Zhao et al. 2010; Tai et al. 2011a). As a kind

Responsible editor: Roland Kallenborn

Lei Xiang and Xiao-Lian Wu contributed equally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s11356-016-6493-8) contains supplementary material,which is available to authorized users.

* Quan-Ying CaiCai_quanying@yahoo.com

* Ce-Hui Motchmo@jnu.edu.cn

1 Guangdong Provincial Research Center for Environment PollutionControl and Remediation Engineering Materials, School ofEnvironment, Jinan University, Guangzhou 510632, People’sRepublic of China

Environ Sci Pollut Res (2016) 23:13984–13995DOI 10.1007/s11356-016-6493-8

http:/dx.doi.org/10.1007/s11356-016-6493-8http://crossmark.crossref.org/dialog/?doi=10.1007/s11356-016-6493-8&domain=pdf

of organic manure, livestock waste has been widely applied toagricultural land, and the amount of antibiotics entering soilvia manure application has been shown to be even higher thanpesticides (Haller et al. 2002; Aga et al. 2005). Some antibi-otics and their metabolites are still biologically active and canresult in severe environmental problems once they enter theenvironment (Zhou et al. 2006). Moreover, antibiotic pollut-ants are different from other organic pollutants such as poly-cyclic aromatic hydrocarbons (PAHs), because they are hy-drophilic and tend to be absorbed and accumulated by vege-tables and other crops grown in contaminated soil (Kumaret al. 2005; Dolliver et al. 2007). Therefore, antibiotic residuescan lead to serious environmental problems, includingdamage to human health and ecological environment.

In recent years, some studies have investigated the occur-rence of antibiotics in soils (Hamscher et al. 2002; Herklotzet al. 2010; Hu et al. 2010; Eggen et al. 2011; Li et al. 2011;Luo et al. 2011; Liu and Wong 2013). For example, Ji et al.(2012) reported that the concentrations of chloramphenicol,sulfonamides, and tetracyclines in agricultural soils adjacentto feedlots in Shanghai, China, were 3.27–33.4 mg/kg, whilethe concentrations of oxytetracycline antibiotics in agricultur-al soils generally ranged from 10 to 1000 μg/kg (Brambillaet al. 2007; Zhang et al. 2008; Karci and Balcioglu 2009). Ourprevious study showed that quinolones, tetracycline, andsulfamethoxazole were detected in more than 94 % of soilsamples from different types of vegetable farms within thePearl River Delta region, South China (In China, vegetablefarms can be classified as conventional, pollution-free, green-food, and organic farmlands; their detailed differences arepresented in Supplementary Material) (Li et al. 2011).Furthermore, antibiotics could be taken up by various vegeta-bles if they were planted in soil contaminated by antibiotics(Hu et al. 2010). The composition and concentrations of anti-biotics in soil are related to the vegetable species, and thehighest concentrations were observed in vegetable fields affil-iated with livestock farms (Li et al. 2011). Nevertheless, Liet al. (2011) did not investigate the occurrence of tetracyclinesin the soils from organic vegetable farms, and did not distin-guish between greenhouse and open-field soil, and especiallydid not evaluate the ecological risk of antibiotics in soil.

Along with social progress, the demand for safer food hasresulted in an increasing desire for organic food products,which are produced without the use of chemical fertilizersand pesticides. In 2009, the global turnover in organic foodwas almost 55 billion US dollars, and the area under organicfarming in Europe accounted for 4.7 % of the total agriculturalarea (IFOAM EU Group and FIBL 2011). Organic farmingavoids the use of synthetic pesticides and chemical fertilizersto reduce the potential contamination of food with chemicalresidues and is often perceived to have generally beneficialimpacts on the environment compared to conventional farm-ing (Tuomisto et al. 2012). However, some toxic organic

pollutants [e.g., PAHs and polychlorinated biphenyls(PCBs)] are frequently detected in organically farmed vegeta-bles and soil (Zohair et al. 2006). In organic vegetable farms,much more organic fertilizers including manure are appliedcompared to conventional farms (Williams and Hedlund2013). The levels of antibiotic residues in soils fertilized withmanure are generally assumed to be higher than in soils fer-tilized with chemicals and other organic fertilizers. Thus, thepotential residue of antibiotics to be present in the products oforganic vegetable farms fertilized with manure is an issue thatrequires further investigation.

Nowadays, very few studies have investigated the occur-rence of antibiotics in the soils of organic farms (Hu et al.2010). Most existing studies have focused on the residues ofantibiotics in the soils of conventional farms, or other areasmainly located in the mid–high latitudes (Karci and Balcioglu2009; Hu et al. 2010; Leal et al. 2012; Xie et al. 2012; Li et al.2013a). Unlike the areas considered in those studies,Guangzhou, the capital city of Guangdong Province, SouthChina, is located in a subtropical region with a higher temper-ature and higher relative humidity. The higher level of mois-ture, temperature, and microbial activity could enhance thetransport, sorption, and degradation of antibiotics in manureand manure-amended soil (Otker and Akmehmet-Balcioglu,2005; Wang et al. 2006; Stoob et al. 2007), which might resultin variations in the levels of antibiotic residues and their envi-ronmental behavior in the soils. Therefore, the main purposesof this study were to investigate the residue levels of tetracy-cline antibiotics in the soil of organic vegetable farms inGuangzhou, South China; to investigate the distribution pat-tern of tetracycline antibiotics under different conditions (openfield and greenhouse); and to assess the potential ecotoxico-logical risk of tetracyclines in soil.

Experimental section

Chemicals and materials

Four tetracycline antibiotics [chlortetracycline (CTC), tetracy-cline (TC), oxytetracycline (OTC), and doxycycline (DC)]were purchased from National Institute for the Control ofPharmaceutical Products (purities >96 %, Beijing, China).High-performance liquid chromatography (HPLC)-grademethanol and acetonitrile were obtained from Sigma-Aldrich(St. Louis, MO, USA). All other reagents were of analyticalgrade. Ultrapure water was used throughout the experiment.

Individual stock solutions of tetracycline antibiotics wereprepared by dissolving 0.0100 g of each antibiotic compoundinto 100 mL of an acetonitrile–water mixture solution (20/80,v/v) containing 1‰ formic acid. All stock solutions werestored at 4 °C in the dark. Working standard solutions were

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prepared by diluting the stock solutions with the acetonitrile–water mixture solution (20/80, v/v) immediately before use.

An EDTA–McIlvaine buffer solution was prepared by dis-solving 27.5 g of Na2HPO4, 37.2 g of Na2EDTA, and 12.9 gof citric acid in 1.0 L of water (pH=4.0). The extraction bufferwas prepared by mixing the EDTA–McIlvaine buffer andmethanol (50/50, v/v).

Sample collection

Guangzhou, a subtropical city, is located in GuangdongProvince, South China. Because of the high temperatureand humidity, three or more batches of vegetables arecultivated annually in this region. From 1996 to 2004,the area of grain sown in Guangzhou decreased by48 %, while that used to grow vegetables increased by60 % (Soil and fertilizer station of Guangdong Province2007). The area of organic vegetable farms inGuangzhou was about 1916 ha. In this study, five rep-resentative organic vegetable farms (referred to as PY,CH, HL, QX, and XA in Fig. 1 and Table S1, BS^indicates the Supplementary Information) were selectedaccording to their geographic location, type of cultiva-tion, scale, and the surrounding environment. The farmswere representative of organic vegetable farms inGuangzhou, South China.

The areas of the five selected farms were between 13.3 and1000.5 ha. In these farms, more than 50 vegetable speciesincluding leaf vegetables, melon or fruit vegetables, and rootor stem vegetables were cultivated, and the agricultural prod-ucts were exported to Japan, Canada, the USA, Europe, HongKong, and other countries and regions. The soils were irrigat-ed with groundwater and fertilized only with commercial or-ganic fertilizer and animal manures (e.g., poultry manure,chicken manure). No synthetic pesticides or chemical fertil-izers were used in the soils of the five selected farms. Due tohigh concentrations of tetracycline antibiotics frequently de-tected in animal manures in China (Supplementary MaterialTable S2) especially in the studied area (Guo et al. 2011), it isspeculated that the manure fertilizers used in the five selectedfarms are the major sources of antibiotics in the soils.

Soil samples were collected from the five farms inNovember 2011. Following the technical guidance for envi-ronmental monitoring, the soil was sampled avoiding the veg-etable field edges, crop roots, and any sites that were justfertilized. In each farm, the sampling sites were selected ac-cording to the vegetable species planted (which could be har-vested at that time). Topsoil samples (depth 0–20 cm) werecollected with a stainless steel spade. Six to eight soil subsam-ples were collected randomly from the sites where each veg-etable species was cultivated. These subsamples were fullymixed to make a composite sample. The soil samples were

Fig. 1 The location of the five organic vegetable farms investigated in Guangzhou

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placed into pre-cleaned brown glass bottles and transferred tothe laboratory as soon as possible. In total, 69 soil sampleswere collected, of which 18were from greenhouse and the restwere from open fields. The soil samples were freeze-dried(Heto Power Dry LL3000; Thermo Scientific, Waltham,MA, USA) and sieved (1 mm) before analysis. The mainphysicochemical properties of soil were measured, and theresults were as follows: 15.1±0.47 g/kg (dry weight) of or-ganic matter, 0.98 ± 0.06 g/kg of total nitrogen (N), 0.83±0.03 g/kg of total phosphorus (P), 20.7±1.01 g/kg of totalpotassium (K), and 4.69±0.21 cmol/kg of cation exchangecapacity.

Sample extraction and cleanup

The extraction and cleanup of soil samples followed themethod developed by Li et al. (2011) with some modifi-cations. A 1.00 g portion of each soil sample was placedin a centrifuge tube containing a 5 mL mixture of EDTA–McIlvaine buffer and methanol (50/50, v/v). The centri-fuge tubes were vortexed (XW-80A, Haimen, China) for1 min and were then extracted three times in an ultrasonicbath (KQ-250E, Kunsan, China) at room temperature for15 min. The extracts were then centrifuged at 4500 r/minfor 10 min. The supernatants were collected into glassbottles and concentrated to several milliliters using a ro-tary evaporator (RE-2000, Shanghai, China). The extractswere concentrated and purified further by solid-phase ex-traction (SPE) using HLB cartridges (3 mL/60 mg,Waters, Milford, MA, USA). The HLB cartridges werepreconditioned sequentially with 6 mL of methanol and6 mL of ultrapure water before the samples were extract-ed. The concentrated supernatants were then passedthrough HLB cartridges. The HLB cartridges were thenrinsed with 6 mL of ultrapure water and vacuum-dried(SHZ-CD, Henan, China) for 10 min. The HLB cartridgeswere eluted twice with 3 mL of methanol. The analyteswere collected into 10-mL glass vials, reduced to neardryness under a nitrogen flow (KL512J, Beijing, China),dissolved in the acetonitrile–water mixture solution (20/

80, v/v) to a final volume of 1 mL, and filtered through0.22-μm syringe filters (Tianjin, China) prior to analysis.

HPLC-MS/MS analysis

Tetracycline antibiotics were analyzed using an HPLC–electrospray ionization tandem mass spectrometry (HPLC-MS/MS) system, following the methods described by Pailleret al. (2009) and Wei et al. (2011) with some modifications.An Alliance 1100 HPLC device (Agilent, Santa Clara, CA,USA) was equipped with a detector with an electrospray ion-ization (ESI) source. The separation of the compounds wasperformed with an Eclipse XDB-C18 column (2.1×150 mm;Agilent, USA). The column temperature was set at 20 °C, andthe injection volume was 5 μL. High-purity water with 0.1 %formic acid was used as mobile phase A, and acetonitrile with0.1 % formic acid was used as mobile phase B, with isocraticconditions set as follows: 0 min 80 % A, 12 min 80 % A. Forthe MS detection, the instrument was operated in positive ion(ESI+) mode for multiple reaction monitoring. Thedesolvation temperature was adjusted to 400 °C, source tem-perature at 120 °C, ion source voltage at 5.5 KV, gas curtaingas at 15.00 Pa, dry gas pressure at 60.0 Pa, and atomizing airpressure at 50.0 Pa. Other mass spectrum conditions were setas shown in Supplementary Table S3.

Method validation

A preliminary study was conducted by analyzing methodblanks to assess the levels of background contamination inthe laboratory. No target compound (i.e., TC, DC, OTC,CTC) was detected in blank samples. Spiked blanks (solventspiked with standards), spiked soil duplicates, and sampleduplicates were routinely analyzed along with each batch ofsoil samples (n=10). Briefly, four tetracycline compounds instandard solution (100 μg/L) were spiked in the mixed solu-tion of EDTA–McIlvaine buffer and methanol (50/50, v/v) orthe soil samples to be extracted, and purified along with othersoil samples. Their average recoveries varied from 72.2 %(TC) to 109.6 % (DC) for spiked blanks and from 63.5 %

Table 1 Concentrations of fourantibiotics in soils from organicvegetable farms (μg/kg, dryweight)

Compounds Maximum Minimum Average Median Detectionfrequency(%)

OTC 31.85 0.04 2.38± 4.63 1.25 100

TC 25.66 0.16 2.67± 4.24 1.10 100

CTC 161.5 0.29 14.50 ± 29.47 5.35 100

DC 184.8 0.87 21.87 ± 32.51 11.36 100

∑TCs 304.7 2.32 40.62 ± 59.64 22.13 100

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(TC) to 93.8 % (DC) for spiked soils, and the relative standarddeviation (RSD) for all analytes was < 10 %. Calibrationcurves of the targeted antibiotics were constructed by injectingmixed standard solutions for quantification. Calibration stan-dards ranging from 0.1 to 100 μg/L with seven points (0.1,0.5, 1, 5, 10, 50, and 100 μg/L) were analyzed by HPLC-MS/MS. The correlation coefficients (R2) of the calibration curvewere>0.999. The limit of detection (LOD) based on a signal-to-noise ratio of three ranged from 0.006 to 0.009 μg/kg. Thelimit of quantification (LOQ) based on a signal-to-noise ratioof 10 ranged from 0.020 to 0.033 μg/kg. The relative standarddeviation (RSD) of parallel samples was

values of the four antibiotics in this study were relativelylower.

Our previous studies have investigated the occurrence ofdifferent antibiotics in the soils of vegetable farms within thePearl River Delta region, South China (Li et al. 2011; Tai et al.2011b). For example, Li et al. (2011) determined three tetra-cycline compounds (OTC, TC, and CTC) in the soils from 21vegetable farms (including conventional, pollution-free, andgreen-food vegetable farms) and their mean concentrationsvaried from 9.6 to 44.1 μg/kg. These levels were significantlyhigher than those recorded in this study. The average concen-trations of TC, CTC, and DC in the soil of a conventionalvegetable farm fertilized chronically with manure were 1.32,5.13, and 5.45 μg/kg, respectively Tai et al. (2011c) whichwere lower than those in the present study, but OTC concen-tration (8.95 μg/kg) was higher compared with the presentstudy. The distribution pattern of tetracycline antibiotics inthe soils of different vegetable farms varied considerably with-in the same region.

The differences between the levels in various regions wereattributed to variations in fertilization practice and the degra-dation of antibiotics in soil (Hu et al. 2010). In Guangzhou,three or more batches of vegetables are planted each year, andthe amount of fertilization applied to agricultural fields is sig-nificantly higher than the global average level (Zeng et al.2012). Generally, in conventional farms, this is applied aseither chemical fertilizers or a mixture of chemical fertilizersand manure, while in organic farms, only organic fertilizers,including manure, are applied (Williams and Hedlund 2013).As described before, animal manure generally contains highconcentrations of various kinds of antibiotics (Hu et al. 2010;Zhao et al. 2010; Tai et al. 2011a; Zhou et al. 2013). Forexample, tetracycline, quinolone, and sulfonamide antibioticshave been detected in swine, cattle, and chicken manures fromsouthern China’s Guangdong Province (Guo et al. 2011; Taiet al. 2011b), and the average concentrations of TC and OTCin the swine and chicken manures of Guangzhou city wassignificantly higher than those reported from the other eightprovinces of China (Zhao et al. 2010; Guo et al. 2011; Zhouet al. 2013). Generally, the organic farms were fertilized withonly commercial organic fertilizers and animal manures,which could be the major sources of antibiotics in the soils(Hu et al. 2010). As showed in Supplementary MaterialTable S2, the TC concentrations (860–326150 μg/kg) in themanures from Guangzhou were comparable with or evenhigher than those from Tianjin (5300–183500 μg/kg).Furthermore, the cultivated vegetables (e.g., leaf and tubervegetable) in the organic vegetable farms in Tianjin (Huet al. 2010) and Guangzhou (the present study) were similar.However, the TC concentrations (0.16–184.8 μg/kg) in thesoils from the organic farms in Guangzhou were lower thanthose in Tianjin (33.1–2683μg/kg, Hu et al. 2010). This mightbe attributed to a higher annual average temperature (26.5 °C)

and relative humidity (77 %) in Guangzhou (located in south-ern China with subtropical marine climate) than Tianjin (lo-cated in northern China with annual average temperature17.9 °C and relative humidity 54 %), because increasing tem-perature and moisture could greatly accelerate the activity ofdegrading microorganisms and the antibiotic degradation inmanure (Otker and Akmehmet-Balcioglu 2005; Wang et al.2006; Stoob et al. 2007; Wang and Yates 2008).

Variation of tetracycline levels in the soils of differentorganic vegetable farms

The concentrations of four tetracycline compounds variedgreatly between the soils of different vegetable farms(Fig. 2c). The highest average ∑TCs (119.6 μg/kg) was ob-served in farm HL, which was more than twice higher than inthe other farms. The lowest average ∑TCs was found in farmCH (12.50 μg/kg). The composition of individual antibioticsin soils also varied among the different farms. DC and CTCwere the dominant compounds in four of the organic vegeta-ble farms (PY, HL, QX, and XA), and they contributed 76.1—94.7 % to the ∑TCs. The soil of farm CH was dominated byCTC and OTC (accounting for 77.8 % of the ∑TCs). Thedistribution pattern of the different compounds investigatedin this study was different from that in the organic vegetablefarms of Tianjin, northern China, where OTCwas the predom-inant antibiotic residue (Hu et al. 2010).

These results can be attributed partly to the differences inmanure types fertilized, the tetracycline composition in ma-nures, and the fertilization history. For example, CTC was thepredominant tetracycline antibiotic found in feces samplesfrom swine and dairy cattle farms in southern China’sGuangxi Province (Zhou et al. 2013), while in the other eightprovinces of China, pig and cow dungwas dominated byOTCand CTC, and chicken dung was dominated by DC (Zhaoet al. 2010). In this study, farm CH was fertilized with swinemanure and commercial organic fertilizers, and farm HL wasfertilized with chicken manure, while commercial organic fer-tilizers were applied in the other farms (SupplementaryTable S1). The type of fertilizers and the composition of tet-racyclines in the manures could affect the distribution of res-idues in soil (Hu et al. 2010).

Additionally, the fertilization history could also influ-ence the residual levels of organic pollutants in soil. Forexample, the residual levels of TC and CTC increased insoil fertilized continuously with liquid manure (Hamscheret al. 2002). However, the residual concentrations of 16PAHs in soils with long-term fertilization of chemical fer-tilizer plus swine manure were significantly lower than insoils with just chemical fertilizer or straw applied, as wellas the control (without fertilization) (Han et al. 2009). Ourprevious study (Tai et al. 2011b) showed that levelsof residual ∑TCs varied from 1.35 to 22.5 μg/kg (mean:

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7.35 μg/kg) in the soil of a pollution-free vegetable farmchronically fertilized with manures in a subtropical area.These levels were considerably lower than those reportedby other researchers (Hamscher et al. 2002; (Brambillaet al. 2007; Karci and Balcioglu 2009) (Zhang et al.2008)). Of the five farms investigated, farm PY has thelongest cultivation history having being established in1994. Farm QX was set up in 2000, while farm CH wasinitiated in 2003. Long-term fertilization with commercialorganic fertilizers or manures could increase the level ofsoil organic matter Li et al. (2013b) and correspondinglyincrease the microbial composition and diversity(Chaudhry et al. 2012; Wu et al. 2013a), which wouldenhance the degradation of organic pollutants. Thus, tet-racycline residues in the soils investigated here, as well asorganic vegetable farms in Tianjin, northern China (Huet al. 2010), were lower than those in the soils of con-ventional farms (Hamscher et al. 2002; Zhang et al. 2008;Li et al. 2011). The concentrations of tetracycline residuesin the soi ls were also affected by degradat ion.Conventional farm management results in a lower activityof soil microorganisms compared with organic farm man-agement (Ge et al. 2010), and thus more degradation ofantibiotics is likely to occur in the soil of an organic farm.The half-life of CTC degradation in different soils (27.6–30.0 days) was longer than TC (20.9–21.7 days) (Li et al.2010), which could result in higher residual levels of CTCthan TC in soil (Table 1).

In this study, the concentrations of tetracyclines in soilsgrowing different vegetable species were also investigat-ed. In Guangzhou, three or more batches of vegetables arecultivated each year. Based on the vegetable types, theplanting models can be classified into Bleaf–leaf–fruitvegetable,^ Bleaf–leaf–melon vegetable,^ Brhizome–mel-on–leaf vegetable,^ and Brhizome–leaf–melon vegetable.^Tetracycline residues in the soils where different vegeta-bles were grown varied greatly, particularly in farm PY(Fig. 3). In the open field of farm PY, higher concentra-tions (∑TCs ranging from 176.8 to 238.2 μg/kg) wereobserved in the soils growing milk Chinese cabbage andonion; conversely, lower concentrations (

higher than in soils growing celery and rape. Moreover,the enhanced dissipation of organic pollutants inrhizospheric soils by various plants was different (Moet al. 2008; Cheema et al. 2010; Li et al. 2013b). All thesefactors might lead to variations in the concentrations ofantibiotics in soils.

Distributions of antibiotics in the soils of open fieldsand greenhouses

The ∑TCs in open-field soil ranged from 2.32 to 304.7 μg/kg(mean: 46.0 μg/kg), while the∑TCs in greenhouse soil variedfrom 4.33 to 83.2 μg/kg (mean: 20.9 μg/kg). The averageconcentrations of individual compounds or ∑TCs in open-field soils were significantly higher (by 1.64–4.06 times) thanin greenhouse soils (Fig. 4). DC and CTC were the dominantcompounds in both greenhouse and open-field soils.

Some researchers have investigated the distributionvariation of heavy metals and pesticides in open-fieldand greenhouse soils, with results indicating that the con-centrations of heavy metals [including chromium (Cr),nickel (Ni), copper (Cu), arsenic (As), cadmium (Cd),and zinc (Zn)] and pesticides in greenhouse soils werehigher than in open-field soils (Li et al. 2008; Baiet al. 2010; Wu et al. 2013a). However, very few studieshave investigated the distribution of antibiotic residues insoils under different cultivation conditions (Maia et al.2009; Li et al. 2013b). Maia et al. (2009) reported noobvious difference in the levels of tetracycline residuesbetween greenhouse and open-field soil (because the tet-racycline was not derived from manure, but as an insec-ticide sprayed on tomatoes) (Maia et al. 2009). The dis-tribution pattern of tetracycline antibiotics in this studywas different from that of heavy metals and pesticides inthe soils of greenhouses and open fields. The phenome-non mentioned above may be related to fertilization con-ditions and the environmental behavior of pollutants in

greenhouse and open-field soils. Some studies have re-ported that the use of fertilizers (including manure) andpesticides is different between greenhouse and open field(Marucci et al. 2011; Zhang et al. 2012; Li et al. 2013a),with higher organic carbon, total nitrogen, soluble organicnitrogen, and cation exchange capacity in greenhouse soilthan in open-field soil (Ge et al. 2010; Zhang et al. 2012;Wang et al. 2013). Moreover, the soil activities (includingmicrobial biomass carbon or nitrogen, sucrase and alka-line phosphatase activities) in a greenhouse have beenshown to be greater than in an open field (Ge et al.2010; Wu et al. 2013a). All of these factors could causedifferences in the loss and retention of water, nutrients,pesticides, and antibiotics in soils between greenhouseand open field (Marucci et al. 2011). For example, Wuet al . (2013b) reported that the dissipation andenantioselective degradation of paclobutrazol anduniconazole differed in greenhouse soil compared to thesoi l f rom an open fie ld in southeastern China.Furthermore, the soil temperature in greenhouse is signif-icantly higher than in open field. The higher temperaturemay increase the activity of soil microorganisms, and cor-respondingly accelerate the degradation of antibiotics insoil (Wang and Yates 2008).

Ecotoxicological risk assessment

The ecotoxicological risk of contaminants in the environmentcan be evaluated on the basis of risk quotient values (RQs),which can be calculated through the measured environmentalconcentration (MEC) or predicted environmental concentra-tion (PEC) of contaminants in the media, divided by the pre-dicted no-effect concentration (PNEC) (EC, 2003; Martinet al. 2012).

According to the European technical guidance documenton risk assessment (European Commission, 2003), PNECvalues are derived from acute toxicity or short-term data (le-thal concentration, LC; effect concentration, EC; and non-observed effect concentration, NOEC) divided by an assess-ment factor of 1000 (Baguer et al. 2000; Fatta-Kassinos et al.2011; Martin et al. 2012; Zhang et al. 2013).

Recently, many studies have assessed the ecotoxicolog-ical risk of pharmaceuticals and antibiotics in the environ-ment (Fatta-Kassinos et al. 2011; Martin et al. 2012;Zhang et al. 2013). However, these studies have mainlyfocused on the risk to the aquatic environment, and veryfew studies have reported the risks to the terrestrial com-partment (particularly to the soil) (Baguer et al. 2000;Fatta-Kassinos et al. 2011; Martin et al. 2012). Thus,PNECsoil values were estimated from PNECwater valuesapplying the equilibrium partition approach suggested bythe European Commission (2003) (Martin et al .2012; Baguer et al. 2000; Fatta-Kassinos et al. 2011;

Fig. 4 Average concentrations of tetracycline antibiotics in greenhouseand open-field soils

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Martin et al. 2012; Baguer et al. 2000; Fatta-Kassinoset al. 2011; Martin et al. 2012):

PNECsoil ¼ PNECwater � Kd; ð1Þ

where Kd is the solid–water partition coefficient. PNECwatervalues were calculated based on the lowest acute toxicity datareported in the literature and an assessment factor of 1000which takes inter-species variations into account (Martinet al. 2012).

In this study, the acute or chronic toxicity data of the fourtetracycline antibiotics using different species were collectedfrom the literature and are presented in SupplementaryTable S4. The toxicity data in presented in bold are for themost sensitive species among those most widely used in tox-icity tests.

The PNECwater values in Table 2 were calculated from thetoxicity data shown in bold. PNECsoil values were estimatedfrom PNECwater values using equation (1) and by taking intoaccount the soil–water Kd values of the tetracycline com-pounds (Pils and Laird 2007; Teixido et al. 2012). The calcu-lated PNECsoil values are shown in Table 2. The RQs for eachtetracycline antibiotic were calculated using the MEC for or-ganic vegetable farm soils (Table 1) and the PNECsoil values(Table 2), and the final RQ values are presented in Fig. 5.

RQ values were categorized into three risk levels: low risk(RQ values 0.01–0.1), medium risk (0.1–1), and high risk(RQ>1) (Martin et al. 2012; Zhang et al. 2013). As shownin Fig. 5, only DC posed a high risk, and the proportion of thesamples causing medium risk and high risk due to DC were55.2 and 44.3 %, respectively. Levels of OTC in 3.0 % of thesamples posed a medium risk, while a low risk to algae oc-curred in 53.7% of the samples. Both TC and CTC posed onlya low risk.

Nevertheless, it should be noted that risk evaluation in thisstudy was based on the toxicity data of individual compoundusing bacteria and algae as target organisms, and thus the risklevels might be over- or underestimated. Because all four ofthe tetracycline antibiotics were detected in soil (Table 1), acombined toxicity effect may exist (Zhu et al. 2013), but this

was not considered here. On the other hand, Baguer et al.(2000) reported that the lowest observed effect concentrationon soil fauna (including earthworms, springtails, andenchytraeids) was 3000 mg/kg of OTC, and in many cases,no effect was observed even at the highest test concentration5000 mg/kg. These results suggest that no risk was posed byOTC in this study because OTC concentrations in the soils

Table 2 Predicted no-effect concentrations (PNECs) and the most sensitive species

Compound Species Toxicity Toxicity data(mg/L)

PNECwater (μg/L) LogKda PNECsoil (μg/kg) Reference

OTC Algae (Pseudokirchneriella subcapitata) growth72 h

EC50= 0.17 0.17 2.7 85.2 Isidori et al. (2005)

TC Algae (Pseudokirchneriella subcapitata) growth72 h

EC50= 1.0 1.0 2.8 631 Yang et al. (2008)

CTC Algae (Pseudokirchneriella subcapitata) growth72 h

EC50= 1.8 1.8 3.1 2266 Park and Choi(2008)

DC Bacteria (B. subtilis) 24 h EC50= 0.009 0.009 3.0 9.0 Suda et al. (2012)

a Data from Pils and Laird (2007) and Teixido et al. (2012)

Fig. 5 The calculated risk quotients (a) and percentages (b) fortetracycline antibiotics detected in the soils of different vegetable farms

13992 Environ Sci Pollut Res (2016) 23:13984–13995

generally ranged from μg/kg levels to several mg/kg (Zhanget al. 2008; Karci and Balcioglu 2009; Hu et al. 2010; Li et al.2011).

However, it should be pointed out that although tetracy-cline antibiotics did not pose as high risk as other organiccontaminants (e.g., PAHs) (Agerso et al. 2006; Man et al.2013), the tetracycline residues could promote the occurrenceof tetracycline-resistant bacteria (Agerso et al. 2006). In or-ganic vegetable farms, long-term application of manuresmight lead to the development of TC resistance in soil bacte-ria. Agerso et al. (2006) reported that the tetracycline resis-tance gene tet(M) could be detected in soil where pig manureslurry had been applied, even when the initial concentrationsof CTC and OTC were only 12.8±1.35 and 3.24±1.65 μg/kg, respectively. Moreover, the level of tet(M) was higher thanin microcosms with the addition of Enterococcus faecalisCG110 (containing the tetracycline-resistant gene tet(M)) orE. faecalis CG110 suspended in pig manure slurry (Agersoet al. 2006). Further research to develop new risk assessmentmethods that combine the RQ values with the tetracyclineresistance gene, e.g., tet(M) is essential.

Conclusions

This study demonstrated that tetracycline antibiotics were fre-quently present in the soil of organic vegetable farms inGuangzhou, with DC and CTC being the dominant com-pounds. However, lower levels of tetracycline residues in soilsfertilized with manures were found in subtropical Guangzhouthan in other studied regions, which might be attributed to thehigh levels of moisture, temperature, and microbial activity inGuangzhou. Risk assessment based on calculated RQ valuesindicated that tetracycline antibiotics in soils posed a low risk(except DC). Further study should be conducted to investigatethe human expose risk to tetracycline antibiotics via organicvegetables and to elucidate the level of tetracycline resistancein organic vegetable farms.

Acknowledgments This work was supported by the National NaturalScience Foundation of China (Nos. 41173101, 41273113, 41301337,41573093), the National Natural Science Foundation of China andGuangdong Province Government Natural Science Joint Foundation(U1501233), and the High-Level Talents Program of GuangdongHigher Education Institutions.

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Occurrence...AbstractIntroductionExperimental sectionChemicals and materialsSample collectionSample extraction and cleanupHPLC-MS/MS analysisMethod validation

Results and discussionOccurrence of tetracycline antibiotics in the soils of organic vegetable farmsVariation of tetracycline levels in the soils of different organic vegetable farmsDistributions of antibiotics in the soils of open fields and greenhousesEcotoxicological risk assessment

ConclusionsReferences